RESEARCH ARTICLE

Alpha Adrenergic Induction of Transport of Lysosomal Enzyme across the Blood-Brain Barrier Akihiko Urayama1, Shinya Dohgu2, Sandra M. Robinson3, William S. Sly4, Jeffery H. Grubb4, William A Banks5,6* 1 Department of Neurology, University of Texas Medical School at Houston, Houston, TX, United States of America, 2 Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka, Japan, 3 Division of Geriatric Medicine, Department of Internal Medicine, Saint Louis University, St. Louis, MO, United States of America, 4 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, United States of America, 5 Geriatric Research, Education, and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, WA, United States of America, 6 Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Washington School of Medicine, Seattle, WA, United States of America * [email protected] OPEN ACCESS Citation: Urayama A, Dohgu S, Robinson SM, Sly WS, Grubb JH, Banks WA (2015) Alpha Adrenergic Induction of Transport of Lysosomal Enzyme across the Blood-Brain Barrier. PLoS ONE 10(11): e0142347. doi:10.1371/journal.pone.0142347 Editor: Mária A. Deli, Hungarian Academy of Sciences, HUNGARY Received: August 19, 2015 Accepted: October 20, 2015 Published: November 6, 2015 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Abstract The impermeability of the adult blood-brain barrier (BBB) to lysosomal enzymes impedes the ability to treat the central nervous system manifestations of lysosomal storage diseases. Here, we found that simultaneous stimulation of the alpha1 and alpha2 adrenoreceptor restores in adult mice the high rate of transport for the lysosomal enzyme P-GUS that is seen in neonates but lost with development. Beta adrenergics, other monoamines, and acetylcholine did not restore this transport. A high dose (500 microg/mouse) of clonidine, a strong alpha2 and weak alpha1 agonist, was able to act as monotherapy in the stimulation of P-GUS transport. Neither use of alpha1 plus alpha2 agonists nor the high dose clonidine disrupted the BBB to albumin. In situ brain perfusion and immunohistochemistry studies indicated that adrengerics act on transporters already at the luminal surface of brain endothelial cells. These results show that adrenergic stimulation, including monotherapy with clonidine, could be key for CNS enzyme replacement therapy.

Data Availability Statement: All data are within the paper. Funding: National Institutes of Health (WAB) Ro1 NS081134, Veterans Affairs (WAB) (no number), Shire (WAB; WSS) (no number). The funders had no role in the study, design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following interests: This study was funded in part by an undesignated grant from Shire. There are no patents, products in development or marketed products to declare. This does not alter the authors'

Introduction Lysosomal storage diseases (LSDs) are autosomal resessive disorders characterized by inherited deficiency in lysosomal metabolic activity. The lack of various acid hydrolases constitutes more than 50 different diseases, each disease having a specific deficiency of a lysosomal enzyme. Enzyme replacement therapy (ERT) has been very effective in treating several LSDs, including mucopolysaccharidoses [1, 2]. While ERT by the intravenous route effectively ameliorates abnormal storage in peripheral organs, correcting central nervous system (CNS) storage has been challenging due to the blood-brain barrier (BBB) hampering the entry of lysosomal

PLOS ONE | DOI:10.1371/journal.pone.0142347 November 6, 2015

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Enzymes Cross the BBB

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enzymes from the blood to brain. For this reason, several approaches are being developed to improve enzyme delivery to the brain, including antibody-directed delivery, improved pharmacokinetics, intrathecal delivery, and targeting of brain endothelial cell transporters [3–5]. Brain microvessel endothelial cells composing the BBB retain several non-specific or specific mechanisms for transcellular transport of macromolecules, including the receptor-mediated, adsorptive-mediated, fluid-phase micropinocytosis, and macropinocytosis [6]. Earlier studies showed that circulating macromolecules appeared in micropinocytic vesicles within the endothelial cells, that micropinocytic vesicles fused with lysosomes, and that macromolecules not resistant to lysosomal degradation were not found beyond the vascular endothelial cell linings [7, 8]. These observations suggested that lysosomes were involved in transcellular transport of lysosomal enzymes in brain endothelial cells, just as they are involved in lysosomal enzyme intracellular trafficking in other types of endothelial cells [9]. Another mechanism for macromolecule transport across the BBB is that of macropinocytosis; the existence of this pathway is lately suggested in human brain microvessel endothelial cells [10, 11]. In our prior studies, we found that the cation-independent mannose 6-phosphate (M6P) receptor participates in brain uptake of systemically circulating lysosomal enzymes across the neonatal BBB [12, 13]. Developmental down-regulation of this receptor-mediated uptake mechanism resulted in failure of brain delivery of lysosomal enzyme across the adult BBB. We postulated that the inability of this transport mechanism in the adult BBB is from the loss of cell surface M6P receptor, while the receptor remains in the intracellular pool. Eventually, we found that M6P receptor-mediated transcytosis of lysosomal enzymes across the BBB was restored by epinephrine in adult mice [14], suggesting that the adrenergic effects of epinephrine modify the transcytotic activity mediated through the M6P receptor which participates in the cellular trafficking of lysosomal enzymes. The regulatory mechanisms involved in the re-induction of the M6P receptor transport of lysosomal enzymes across the BBB by epinephrine remain to be elucidated. Currently, there is no direct evidence that epinephrine modulates the activity of the M6P receptor itself. Brain microvessel endothelial cells express both α- and β-adrenoceptors [15]. Both adrenoceptors, including receptor subtypes of each, can initiate the internalization of receptors, inducing their redistribution from the cell surface to cytoplasmic vesicles [16–18]. The biological role of receptor internalization may have a variety of spatio-temporal effects, possibly including effects on the redistribution of endosomal M6P receptors. The present study addresses the regulatory mechanisms by which the adrenergic system modulates the transport of P-GUS across the adult BBB by employing series of receptor agonists and antagonists in vivo.

Materials and Methods Production of PGUS PGUS was produced in overexpressing, cation-independent M6P receptor-deficient mouse L cells as described previously [4]. The enzyme was purified from conditioned media by antihuman GUS mAb affinity column chromatography. PGUS was eluted with 3.5 M MgCl2, then desalted over Bio Gel P6 sizing resin (Bio-Rad, Hercules, CA). The concentration of PGUS was adjusted to 2.5 × 105 units per ml (1 unit = 1 nmol of substrate cleaved per h) and the purified enzyme was stored at -70°C. M6P-specific uptake of the PGUS by human fibroblasts was 185 units per mg/h (data not shown).

Radioactive labeling PGUS was radioactively labeled using the iodobead method (Pierce, Rockford, IL) with [131I] Na (Perkin Elmer, Waltham, MA). The use of a single iodobead allows controlled iodination,

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Enzymes Cross the BBB

so that both enzymatic activity and susceptibility to endocytosis by the M6P receptor are preserved [12–14]. Labeled, active enzyme (I-PGUS) was separated from free iodine on a Sephadex G-10 column. Albumin was labeled with [125I]Na (Perkin Elmer) using the chloramine-T method, and purified (I-Alb) on a column of Sephadex G-10. Each reagent was freshly prepared on the day of the experiment.

Animals Adult male CD-1 mice from our in-house colony were studied at 7–8 weeks of age. The mice had free access to food and water and were maintained on a 12-hour dark/light cycle in a room with controlled temperature. All the animal studies were approved in advance by the St. Louis VA Institutional Animal Care and Use Committee, conducted according to national and international guidelines, and carried out at the VA which is a facility that is approved by the Association for Assessment and Accreditation of Laboratory Animal Care.

Epinephrine Effects on I-PGUS influx and BBB disruption Multiple-time regression analysis was used to measure blood-to-brain uptake of I-PGUS and I-Alb [19, 20]. Disruption of the BBB was measured as an increase in the brain/serum ratio for I-Alb and the unidirectional influx rate of I-PGUS was measured after correction for the I-Alb space. Adult male CD-1 mice were anesthetized with an I.P. injection of urethane (40%) and a 200 μl injection of lactated Ringers solution containing1% BSA and 5.5x105 cpm each of I-PGUS or I-Alb was injected into the jugular vein with or without 40 or 120 nmol/injection of epinephrine. Brain and carotid artery blood samples were obtained 1, 2, 3, 4, 5, 6, 7.5, and 10 min after the iv injection (n = 3-4/time point), arterial blood was collected from the carotid artery and the brain was removed and weighed. Levels of radioactivity in the brain and 50 μl of serum were counted in a gamma counter for 3 minutes. Brain/serum ratios were calculated to yield units of μl/g and plotted against exposure time. The linear portion of the slope of the resulting correlation measures the unidirectional influx rate in units of μl/g-min and the Yintercept measures the distribution space in brain at t = 0 in units of μl/g. The effect of epinephrine on blood clearance and volume of distribution were also measured by plotting the percent of the injected dose found per ml of serum (%Injected dose/ml) against time.

Monoamines, Acetylcholine, and Adrenergic Agents All reagents were purchased from Sigma-Aldrich Chemical Co (St Louis MO) unless otherwise noted. Reagents were dissolved in lactated Ringers solution with 1% by weight of bovine serum albumin unless otherwise noted. Concentrations are given in moles/mouse except when the literature has primarily given doses in g/mouse, in which case moles are given in parentheses. Adult male mice anesthetized with urethane were given an injection into the jugular vein of lactated Ringer’s solution containing 1% BSA and 4x105 cpm of both I-PGUS and I-Alb with or without an agent. For antagonists, 40 nmol/mouse of epinephrine was included in the iv injection. After 10 min, blood was obtained from the carotid artery and the brain removed. Serum derived from the arterial blood and the whole brain were counted in a gamma counter for 3 min and the results expressed as brain/serum ratios in units of μl/g. Acetylcholine and the monoamines dopamine, histamine, and serotonin were tested at the dose of 200 nmol/mouse. General adrenergic receptor antagonists tested were propranolol (general beta) 200 nmol/ mouse, yohimbine (alpha2) 100 nmol/mouse, phentolamine (general alpha) 200 nmol/mouse, and prazocin (alpha1) 100 nmol/mouse. Antagonists that were more selective for their sites were benoxathian (specific alpha1) 250 μg/mouse (628 nmol/mouse) and CGP20712A (specific beta) 250 μg/mouse (420 nmol/mouse). General agonists tested were isoproterenol (general

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beta agonist) 10 nmol/mouse, L-phenylephrine (alpha1) 10 nmol/mouse, and clonidine (alpha2) 10 nmol/mouse. Agonists that were more selective for their sites were UK 14,304 with it and its control tested in 14.9% DMSO (alpha2) 200 μg/mouse (682 nmol/mouse), albuterol (selective beta) 200 μg/mouse (694 nmol/mouse), and cirazoline (alpha1) 100 μg/mouse (463 nmol/mouse) In follow-up studies, cirazoline 100 μg/mouse plus clonidine 200 μg/mouse were also tested and clonidine alone was tested at 50, 200, and 500 μg/mouse.

Immunohistochemistry in brain endothelial cell monolayers Primary culture of mouse brain endothelial cells were isolated from adult male CD-1 mice (8 weeks old) as previously described [21]. Brain microvessel endothelial cells were treated with or without epinephrine (10 μM) in serum-free DMEM/F-12 (Sigma) supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamicin, 2 mM GlutaMAX™-I and 1 ng/mL basic fibroblast growth factor (bFGF; Sigma) for 30 min. After fixation with 3.7% formaldehyde (Sigma) for 10 min at room temperature, they were incubated with anti-M6P receptor antibody (Abcam) in 1% BSA/PBS for overnight at 4°C. Then, they were washed once with PBS, three times with balanced salt solution (130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 4 mM MgCl2, 20 mM HEPES, 5.5 mM glucose, pH 7.4) and once with PBS, and incubated with 20 μg/mL Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen) in 1% BSA/PBS for 1 hr at room temperature. After washing, cells were covered with Vectashield Hard Set mounting medium (Vector Laboratories, Burlingame, CA) and coverslips were applied. Fluorescence was detected with Zeiss Axiovert 40 CFL fluorescent microscope.

In situ transcardiac brain perfusion Adult male CD-1 mice were anesthetized with 40% urethane, the heart exposed, both jugulars severed, and the descending thoracic aorta ligated. A 26-gauge butterfly needle was inserted into the left ventricle of the heart, and the perfusion fluid (7.19 g/liter NaCl, 0.3 g/liter KCl, 0.28 g/liter CaCl2, 2.1 g/liter NaHCO3, 0.16 g/liter KH2PO4, 0.17 g/liter anhydrous MgCl2, 0.99 g/liter d-glucose, and 1% wt/vol BSA), containing 105 cpm each of I-PGUS and I-Alb with or without 1 nmol/ml or 10 nmol/ml of epinephrine was infused at a rate of 2 ml/min for 5 min. The perfusion fluid was freshly prepared each day. After perfusion, the whole brain was removed and weighed. The level of radioactivity was determined in a γ-counter.

Statistical Analysis Means are reported with their standard error terms. Statistical comparisons were made between two groups using Student’s t-test. More than two groups were compared by analysis of variance (ANOVA) with Newman-Keuls used as the post-test. Regression lines were computed by the least squares method and the slopes and intercepts compared using the statistical package in Prism 5.0 (GraphPad, Inc San Diego, CA).

Results Epinephrine on I-PGUS influx and BBB disruption Fig 1 shows the serum level-time profiles (panels A and C) and the brain/serum ratios (panels B and D) of I-albumin and I-PGUS after i.v. injection with epinephrine. Epinephrine at the 120 nmol, but not the 40 nmol/mouse dose, slightly decreased the distribution space for I-Albumin (F(2,61) = 6.3, p

Alpha Adrenergic Induction of Transport of Lysosomal Enzyme across the Blood-Brain Barrier.

The impermeability of the adult blood-brain barrier (BBB) to lysosomal enzymes impedes the ability to treat the central nervous system manifestations ...
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